Boden, A. | Search bioRxiv

bioRxiv2020

Three dimensional parallelized RESOLFT nanoscopy for volumetric live cell imaging

The volumetric architecture of organelles and molecules inside cells can only be investigated with microscopes featuring sufficiently high resolving power in all three spatial dimensions. Current methods suffer from severe limitations when applied to live cell imaging such as long recording times and/or photobleaching. By introducing a novel optical scheme to switch reversibly switchable fluorescent molecules, we demonstrate volumetric nanoscopy of living cells with resolution below 100 nm in 3D, large field of view and minimal illumination intensities (W-kW/cm2).

biophysics↗

bioRxiv2018

Enhanced photon collection enables four dimensional fluorescence nanoscopy of living systems

The theoretically unlimited spatial resolution of fluorescence nanoscopy often comes at the expense of time, contrast and increased dose of energy for recording. Here, we developed MoNaLISA, for Molecular Nanoscale Live Imaging with Sectioning Ability, a nanoscope capable of imaging structures at a scale of 45-65 nm within the entire cell volume at low light intensities (W-kW/cm2). Our approach, based on reversibly switchable fluorescent proteins, features three distinctly modulated illumination patterns crafted and combined in a novel way to gain fluorescence ON-OFF switching cycles and image contrast. By maximizing the detected photon flux MoNaLISA enables prolonged (40-50 frames) and large (50 x 50 m2) recordings at 0.3-1.3 Hz with enhanced optical sectioning ability. We demonstrated the general use of our approach by 4D imaging of organelles and fine structures in epithelial human cells, colonies of mouse embryonic stem cells, brain cells, and organotypic tissues.

biophysics↗